Refractive index detection in microbore column liquid chromatography

Summary An existing commercial refractive index detector was modified for use with microbore column LC systems. The detector utilizes the Fresnel method. The effect of band dispersion and dilution at the detector side is of extreme importance in connection with the miniaturized LC system. In the modified model the original heat-exchanger tube was removed and a stainless steel capillary was used for heat-exchanging. Gaskets having different cell volumes were also examined with respect to band broadening and sensitivity. The detection limit was 10ng for di-n-pentyl phthalate. The examples include the detection of phthalates, alcohols, n-paraffins, and kerosine.     

Enzyme-based biosensor as a selective detection unit in column liquid chromatography

A reagentiess enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was ferinented with Saccharomyces cerevisiae.     

Scintillator-fiber flow-cell radioactivity detector for liquid chromatography

Summary A new type of flow-cell radiation detector for use in liquid chromatography which is packed with aligned scintillator fibers is described. A primary advantage of the fiber packed cell is that light generated by the scintillator is absorbed to a much less extent by the fibers than by the powder scintillator used in conventional flow cells. A detection efficiency of 55% has been obtained for carbon-14 using 0.1-mm diameter hand-pulled glass fibers. Computer modeling has shown that even better results can be obtained by using smaller and more uniform diameter fibers which will allow better packing. The fiber cell also demonstrates back pressures which are a factor of 50 less than the conventional cell and much less susceptibility to absorption of compounds because of its lower surface area. Research sponsored by the Office of Energy Research, U.S. Department of Energy, under contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.     

Determination of zopiclone in plasma using column liquid chromatography with ultraviolet detection.

A reversed-phase liquid chromatographic method with ultraviolet detection for the determination of zopiclone in plasma is described. It is rapid, sensitive, reproducible and linear over a wide range. The method was used to study plasma zopiclone concentrations in a case of acute intoxication after oral ingestion of 300 mg of the drug. The plasma level was 1600 ng/ml 4.5 h after the dose and the elimination half-life was 3.5 h.     

Microbore columns in quantitative column liquid chromatography

Summary Some quantitative implications of the use of microbore columns in column liquid chromatography are investigated. Although for several pumps the flow rate stability at 50 l min^–1 is slightly worse than that of pumps operating at 1 ml min^–1, the quantitative performance with respect to repeatability and reproducibility of response factors equals that of conventional liquid chromatography. Thermostatting is strongly recommended for microbore column operation.     

Determination of naphazoline in rat plasma using column liquid chromatography with ultraviolet detection.

A sensitive (1 ng/mL) and rapid method for the determination of naphazoline in rat plasma is described. Following extraction, the compound is analysed by reversed phase high performance liquid chromatography and ultraviolet detection at 214 nm.     

Determination of retinoids in galenicals by column liquid chromatography with fluorescence and diode-array detection

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.     

Enzyme based detection systems in column liquid chromatography — Extension of selectivity and sensitivity

Summary Enzyme based detection systems in column liquid chromatography are described using systems based on both enzyme reactors in conjunction with a flow through detection device and enzyme electrodes. Optimizations for making detection principles compatible with chromatographic systems and applications made in our laboratories are presented for the analysis of e.g. carbohydrates, amino acids, ethanol and catecholamines. Gel-permeation chromatography and mass spectrometry are used as complementary techniques for qualitative and quantitative analysis of complex samples.     

Fast gas chromatography: packed column solvating gas chromatography versus open tubular column gas chromatography

Packed capillary column solvating gas chromatography (SGC) and open tubular column gas chromatography (GC) were compared with respect to their potentials for fast separations. A recently introduced "universal" peak capacity equation was used to compare the performance of these two methods. The effects of various factors on peak capacity were investigated. Results demonstrate that retention factor and column efficiency are the main factors affecting peak capacity for fast separations. Packed columns produce both high retention factors and high selectivities. While high efficiencies and high peak capacities can be demonstrated by both techniques, open tubular column GC can surpass packed capillary column SGC in both measurements, except for the case of the analysis of simple mixtures in short analysis times, where retention factor and selectivity become important. Practical aspects such as pressure drop and sample capacity are compared for SGC and open tubular column GC. It was found that packed column SGC demonstrates higher sample capacities, but requires much higher column inlet pressures than open tubular column GC. A variety of mobile phases can be used for packed column SGC, which can provide high solvating power for large and polar compounds.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Control of column temperature in reversed-phase liquid chromatography

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.     

Improved liquid chromatography-Online radioactivity detection for metabolite profiling

If very-high-pressure liquid chromatography (VHPLC) is to replace conventional HPLC as the ultimate separation tool for metabolism studies in development, coupling it efficiently with online radioactivity detection (RAD) is needed. We describe the successful combination of VHPLC/RAD, facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity. The sensitivity of (14)C detection was improved by the use of a variable scintillation flow achieved via a simple modification to the classical online radiochemical detection set-up. A modification of the flow-through cell design in which internal diameter of the tubing was reduced further increased the sensitivity and resolution by decreasing peak tailing. The injection of relatively large injection volumes was made possible by the use of columns packed at ultra-high pressure with 2.2 microm particles. Because of the reduced back pressure generated using these larger particle sizes, two 150 mm x 3 mm columns could be coupled, allowing 4-fold larger injection volumes and a 50% increase in theoretical plate number at a similar back pressure compared to a standard 150 mm x 2.1mm Waters UPLC column. The value of the methodology described was demonstrated by the analysis of in vitro and in vivo metabolism samples of (3)H- and (14)C-labeled compounds and compared with conventional radio-HPLC. We have shown that metabolite separation can be achieved with increased efficiency while maintaining a sensitivity comparable to that of conventional HPLC/RAD.     

Analysis of parabens in cosmetics by low pressure liquid chromatography with monolithic column and chemiluminescent detection

This paper presents an application of chromatographic separation based on an ultra-short monolithic column and chemiluminescent detection in an FIA type instrument manifold for the determination of four paraben mixtures: methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP). The separation is achieved in 150 s using two consecutive carriers: first 12% ACN:water that changes 75 s after injection to 27% ACN:water. The detection is based on the oxidation of the hydrolysis product of parabens, p-hydroxybenzoic acid, with Ce(IV) in the presence of Rhodamine 6G which evokes chemiluminescence of sufficient intensity to enable a sensitive determination of these species. After optimization of the variables involved, the analytical method is characterized, displaying the following values for concentration ranges, detection limits and precision, as relative standard deviation at low concentration (0.15 mg l⁻¹)—MP: from 9.9 × 10⁻⁷ to 3.3 × 10⁻⁴ M; 1.9 × 10⁻⁸; 5.6%; EP: from 9.0 × 10⁻⁷ to 3.3 × 10⁻⁴ M; 2.8 × 10⁻⁸; 3.5%; PP: from 8.3 × 10⁻⁷ to 9.9 × 10⁻⁵ M; 2.3 × 10⁻⁸; 4.2%; and BP: from 7.7 × 10⁻⁷ to 9.9 × 10⁻⁵ M; 4.2 × 10⁻⁸ M; 6.2%. The method was applied and validated satisfactorily for the determination of these parabens in cosmetic samples, comparing the results against a liquid chromatography reference method.     

Multiresidue determination of fluoroquinolones in milk by column liquid chromatography with fluorescence and ultraviolet absorbance detection

Column liquid chromatography with fluorescence (FLD) and UV-diode array detection (UV-DAD) was used for the simultaneous determination of ciprofloxacin (CIPRO), enrofloxacin (ENRO), marbofloxacin (MARBO), danofloxacin (DANO) and sarafloxacin (SARA) residues in milk, using norfloxacin (NOR) as internal standard. Two solid-phase extraction (SPE) cartridges, were evaluated for sample clean-up and preconcentration, Strata X, based on a modified styrene-divinylbenzene polymer, and Strata Screen A, a mixed anion exchanger/C8 reversed-phase sorbent. The fluoroquinolones (FQs) were separated on a polar endcapped column (AQUA C18). The recoveries for raw milk spiked with the antibiotics at three concentrations close to the maximum residue limit (MRL), were 80-103% for ENRO, CIPRO and DANO, with relative standard deviations (R.S.D.) lower than 6.6%. SARA recoveries were 70% (R.S.D. = 7%) and values in the order of 95% (R.S.D. = 1.5%) were obtained for MARBO at the MRL level. The quantification limits ranged from 2.4 to l0 ng ml(-1) and are below the MRL established for these drugs by the European Union. The method was successfully applied to the analysis of ENRO and its metabolite CIPRO in an incurred milk sample.     

Quantitative analysis of tylosin by column liquid chromatography

A column liquid chromatographic method suitable for the quality control of tylosin A is described. The determination can be carried out on different C₈ or C₁₈ columns, using a mobile phase containing acetonitrile, 0.2 M tetrabutylammonium hydrogensulphate, 0.2 M phosphoric acid and water. The flow-rate is 1 ml/min and detection is performed at 280 nm. The method shows good selectivity towards the major components tylosin A, B, C and D and demycinosyltylosin. Minor degradation products, mainly observed in solutions, are also separated. The compositions of several standards are compared and results for a number of commercial samples are presented.